Chronic hypoxia, as occurs in many pulmonary diseases, results in vascular myocyte proliferation and pulmonary hypertension. Pulmonary arterial smooth muscle cells (PASMCs) from animals of chronic hypoxia are associated with an elevated [Ca2+]i and altered reactivity to agonists, suggesting alterations in Ca2+ homeostasis intrinsic to PASMCs. It has been postulated that chronic hypoxia suppresses KV channel expression, leading to membrane depolarization, activation of L-type Ca2+ channels, and increase in [Ca2+]i. However, some studies found that inhibition of L-type Ca2+ channels was ineffective in reducing the elevated [Ca2+]i and vasomotor tone, suggesting additional Ca2+ pathway(s) may be involved. Recently, we have identified multiple TRPC channel isoforms (TRP1, TRP3, and TRP6) in rat intra-lobar PASMCs. Semi-quantitative RT-PCR analyses show that TRPC1 and TRPC6 mRNA level were increased in chronic hypoxic PASMCs; functional studies showed significant increase in store- and receptor-operated Ca2+ entry. In addition, we found that local Ca2+ release transients, "Ca2+ sparks", have very robust interactions with IP3-receptors and TRPCs. Both spontaneous and agonist-evoked Ca2+ spark activities were altered in chronic hypoxic PASMCs. Based on these findings, we hypothesis that multiple Ca2+ influx and release pathways are altered by chronic hypoxia; the increase in cation entry via TRPCs, and alterations in SR Ca2+ release processes play major roles in the increase in basal [Ca2+]i and vasomotor tone, and the alterations in vascular reactivity. To test these hypotheses, we will apply a combination of state-of-the-art techniques including whole-cell patch clamp, laser-scanning confocal microscopy, UV-pulse laser flash photolysis, microarray analysis, antisense gene knockout and isolated microvessels to examine (i) changes in gene-expressions of various Ca2+ transporters, and the associated changes in vascular reactivity, (ii) changes in ryanodine- and IP3-receptors dependent Ca2+ release, (iii) alterations in store-operated and receptor-operated Ca2+ entries, and (iv) specific TRPC subtypes responsible for the elevated basal [Ca2+]i in PASMCs and vasomotor tone in pulmonary arteries of chronic hypoxic rats. This project will provide unique information on the subcellular Ca2+ signaling and homeostasis in pulmonary vasculatures in chronic hypoxia-induce pulmonary hypertension.